Calpain is a calcium-dependent protease that plays a significant role in synaptic plasticity, cell motility, and neurodegeneration. Two major calpain isoforms are present in brain, with -calpain (calpain1) requiring micromolar calcium concentrations for activation and m-calpain (calpain2) needing millimolar concentrations. Recent studies in fibroblasts indicate that epidermal growth factor (EGF) can activate m-calpain independently of calcium via mitogen-activated protein kinase (MAPK)-mediated phosphorylation. In neurons, MAPK is activated by both brain-derived neurotrophic factor (BDNF) and EGF. We therefore examined whether these growth factors could activate m-calpain by MAPK-dependent phosphorylation using cultured primary neurons and HEK-TrkB cells, both of which express BDNF and EGF receptors. Calpain activation was monitored by quantitative analysis of spectrin degradation and by a fluorescence resonance energy transfer (FRET)-based assay, which assessed the truncation of a calpain-specific peptide flanked by the FRET fluorophore pair DABCYL and EDANS. In both cell types, BDNF and EGF rapidly elicited calpain activation, which was completely blocked by MAPK and calpain inhibitors. BDNF stimulated m-calpain but not -calpain serine phosphorylation, an effect also blocked by MAPK inhibitors. Remarkably, BDNF-and EGF-induced calpain activation was preferentially localized in dendrites and dendritic spines of hippocampal neurons and was associated with actin polymerization, which was prevented by calpain inhibition. Our results indicate that, in cultured neurons, both BDNF and EGF activate m-calpain by MAPK-mediated phosphorylation. These results strongly support a role for calpain in synaptic plasticity and may explain why m-calpain, although widely expressed in CNS, requires nonphysiological calcium levels for activation.

No abstract

17-␤-Estradiol (E2) is a steroid hormone involved in numerous brainfunctions. E2 regulates synaptic plasticity in part by enhancing NMDA receptor function and spine density in the hippocampus, resulting in increased long-term potentiation and facilitation of learning and memory. As the calcium-dependent neutral protease, calpain, is also involved in these processes, we tested whether E2 could activate calpain and examined the functional consequences of E2-mediated calpain activation in hippocampus. Calpain activity was analyzed by a fluorescence resonance energy transfer (FRET)-based assay that allows both quantitative determination and spatial resolution. E2 rapidly activated calpain in cultured cortical and hippocampal neurons, prominently in dendrites and dendritic spines. E2-induced calpain activation was mediated through mitogen-activated protein kinase (MAPK), as it was completely blocked by MEK inhibitors. It was also calcium-independent, as it was still evident in presence of the calcium chelator, BAPTA-AM. Activation of ER␣ and ER␤ receptors by specific agonists stimulated calpain activity. Finally, the rapid E2-mediated increase in excitability in acute hippocampal slices was prevented by a membrane-permeable calpain inhibitor. Furthermore, E2 treatment of acute hippocampal slices resulted in increased actin polymerization and membrane levels of GluR1 but not GluR2/3 subunits of AMPA receptors; both effects were also blocked by a calpain inhibitor. Our results indicate that E2 rapidly stimulates calpain activity through MAP kinase-mediated phosphorylation, resulting in increased membrane levels of AMPA receptors. These effects could be responsible for E2-mediated increase in neuronal excitability and facilitation of cognitive processes.FRET ͉ hippocampus ͉ synaptic plasticity C alpains are intracellular calcium-dependent neutral cysteine proteases that have been implicated in several brain processes, including synaptic plasticity [e.g., long term potentiation (LTP)] and learning and memory formation (1, 2). To account for the role of calpain in these processes, calpain activity has been proposed to be intricately involved in activity-dependent changes in dendritic spine morphology via truncation of a variety of cytoskeletal proteins, including spectrin, as well as of enzymes participating in regulation of spine morphology and function (3-6). Several studies have indicated that m-calpain could be rapidly activated by MAP kinase-mediated phosphorylation in various cell types (7-9), and we recently reported that a similar process occurs in neurons (10). Moreover, we showed that brain-derived neurotrophic factor (BDNF)-elicited increase in actin polymerization in cultured cortical neurons was blocked by a calpain inhibitor (10). These findings provided a link between several elements proposed to play a critical role in LTP, namely BDNF, calpain, MAP kinase, and actin polymerization.17-␤-Estradiol (E2) is a steroid hormone that is critical for the functions of multiple organs, including the brain. In partic...

Toward identifying a cancer-specific gene signature we applied surprisal analysis to the RNAs expression behavior for a large cohort of breast, lung, ovarian, and prostate carcinoma patients. We characterize the cancer phenotypic state as a shared response of a set of mRNA or microRNAs (miRNAs) in cancer patients versus noncancer controls. The resulting signature is robust with respect to individual patient variability and distinguishes with high fidelity between cancer and noncancer patients. The mRNAs and miRNAs that are implicated in the signature are correlated and are known to contribute to the regulation of cancer-signaling pathways. The miRNA and mRNA networks are common to the noncancer and cancer patients, but the disease modulates the strength of the connectivities. Furthermore, we experimentally assessed the cancer-specific signatures as possible therapeutic targets. Specifically we restructured a single dominant connectivity in the cancerspecific gene network in vitro. We find a deflection from the cancer phenotype, significantly reducing cancer cell proliferation and altering cancer cellular physiology. Our approach is grounded in thermodynamics augmented by information theory. The thermodynamic reasoning is demonstrated to ensure that the derived signature is bias-free and shows that the most significant redistribution of free energy occurs in programming a system between the noncancer and cancer states. This paper introduces a platform that can elucidate miRNA and mRNA behavior on a systems level and provides a comprehensive systematic view of both the energetics of the expression levels of RNAs and of their changes during tumorigenicity. microarray | deep sequencing | network connectivity | biomarker | maximal entropy

For more than 15 years, angiotropism in melanoma has been emphasized as a marker of extravascular migration of tumor cells along the abluminal vascular surface, unveiling an alternative mechanism of tumor spread distinct from intravascular dissemination. This mechanism has been termed extravascular migratory metastasis (EVMM). During EVMM, angiotropic tumor cells migrate in a 'pericytic-like' manner (pericytic mimicry) along the external surfaces of vascular channels, without intravasation. Through this pathway, melanoma cells may spread to nearby or more distant sites. Angiotropism is a prognostic factor predicting risk for metastasis in human melanoma, and a marker of EVMM in several experimental models. Importantly, analogies of EVMM and pericytic mimicry include neural crest cell migration, vasculogenesis and angiogenesis, and recent studies have suggested that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes reminiscent of cancer migration and embryonic/stem cell state transitions. A recent work revealed that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression via angiotropism and migration along the abluminal vascular surface. Finally, recent data using imaging of melanoma cells in a murine model have shown the progression of tumor cells along the vascular surfaces. Taken together, these data provide support for the biological phenomenon of angiotropism and EVMM, which may open promising new strategies for reducing or preventing melanoma metastasis.